This research application focuses on mechanistic studies of three key enzymes in the Wood-Ljungdahl pathway: CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), methyltransferase (MeTr), and the corrinoid iron-sulfur protein (CFeSP). Studies on this system have enriched the areas of microbiology, biochemistry, and metallobiochemistry in revealing the structures of macromolecular channels and previously unknown metal clusters. These studies are providing insight into how these proteins use bioorganometallic intermediates in catalyzing group transfer reactions and C-C and C-S bond formation and cleavage allowing organisms to assimilate carbon dioxide and the toxic gas, CO. This system serves as a paradigm for understanding bioinorganic chemical principles, complex protein-protein interactions, and how proteins coordinate redox reactions with chemical catalysis. The major focus of this application is on multidisciplinary structure-functions studies of the three major classes of Ni-CODHs: the monofunctional CODH, the bifunctional CODH/ACS from acetogenic bacteria, and the bifunctional CODH/ACS (also called acetyl-CoA decarbonylase synthase, ACDS) from aceticlastic methanogens. Because these enzymes play different physiological roles, they are hypothesized to exhibit correspondingly disparate catalytic properties, including different mechanisms of substrate channeling, catalytic biases for CO oxidation/carbon dioxide reduction, and electrochemical properties. The proton transfer network including the acid-base catalyst(s), conserved among all CODH classes, will be further investigated. The early intermediate(s) in the CODH mechanism will be trapped and characterized. The internal electron transfer reaction in the ACS mechanism will be characterized. We will further investigate how the CO channel in CODH/ACS meters the supply of and demand for CO and investigate how the CODH and ACS sites in CODH/ACS are coordinated. We also will determine the crystal structure of the CFeSP- MeTr complex and investigate the proposed role of a major conformational change in the reaction of MeTr with the CFeSP. Studies on PFOR will test a kinetic coupling hypothesis, including identification of a proposed radical species formed by reaction of CoA with the hydroxyethyl-TPP radical. PUBLIC HEALTH RELEVANCE: We are studying the key enzymes in a pathway by which anaerobic microbes assimilate carbon dioxide and carbon monoxide. These studies involve the elucidating new roles of metal ions in biology (metal-carbon bonds, new heterometallic clusters, and nucleophilic metal ions), characterizing novel substrate-derived radical intermediates, and describing channels for gaseous substrates within an enzyme.